Perfusive chromatography

ABSTRACT

Disclosed are chromatography methods and matrix geometries which permit high resolution, high productivity separation of mixtures of solutes, particularly biological materials. The method involves passing fluids through specially designed chromatography matrices at high flow rates. The matrices define first and second interconnected sets of pores and a high surface area for solute interaction in fluid communication with the members of the second set of pores. The first and second sets of pores are embodied, for example, as the interstices among particles and throughpores within the particles. The pores are dimensioned such that, at achievable high fluid flow rates, convective flow occurs in both pore sets, and the convective flow rate exceeds the rate of solute diffusion in the second pore set. This approach couples convective and diffusive mass transport to and from the active surface and permits increases in fluid velocity without the normally expected bandspreading.

This application is a continuation of application Ser. No. 376,885,filed July 6, 1989, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to methods and materials for conducting very highefficiency chromatographic separations, i.e., adsorptive chromatographytechniques characterized by both high resolution and high throughput perunit volume of chromatography matrix. More specifically, the inventionrelates to novel geometries for matrices useful in chromatography,particularly preparative chromatography, and to methods for conductingchromatographic separations at efficiencies heretofor unachieved.

The differences in affinities of individual solutes for a surface basedon charge, hydrophobic/hydrophilic interaction, hydrogen bonding,chelation, immunochemical bonding, and combinations of these effectshave been used to separate mixtures of solutes in chromatographyprocedures for many years. For several decades, liquid chromatography(LC) has dominated the field of analytical separation, and often hasbeen used for laboratory scale preparative separations. Liquidchromatography involves passing a feed mixture over a packed bed ofsorptive particles. Subsequent passage of solutions that modify thechemical environment at the sorbent surface results in selective elutionof sorbed species. Liquid flows through these systems in theinterstitial space among the particles.

The media used for liquid chromatography typically comprises softparticles having a high surface area to volume ratio. Because of theirmany small pores having a mean diameter on the order of a few hundredangstroms (Å) or less, 95% or more of the active surface area is withinthe particles. Such materials have been quite successful, particularlyin separation of relatively small chemical compounds such as organics,but suffer from well-recognized limits of resolution for largermolecules. Liquid chromatography materials also are characterized byoperational constraints based on their geometric, chemical, andmechanical properties. For example, soft LC particles cannot be run atpressure drops exceeding about 50 psi because the porous particles areeasily crushed.

Recently, high performance liquid chromatography (HPLC) has becomepopular, particularly for analytical use. Instead of employing soft,particulate, gel-like materials having mean diameters on the order of100 μm, HPLC typically employs as media 10 to 20 μm rigid porous beadsmade of an inorganic material such as silica or a rigid polymer such asa styrene divinylbenzene copolymer. HPLC allows somewhat faster andhigher resolution separations at the expense of high column operatingpressure drops.

Products emerging from the evolving biotechnology industry present newchallenges for chromatography. Typically, these products are large andlabile proteins having molecular weights within the range of 10⁴ to 10⁶daltons. Such products are purified from mixtures which often containhundreds of contaminating species including cell debris, varioussolutes, nutrient components, DNA, lipids, saccharides, and proteinspecies having similar physicochemical properties. The concentration ofthe protein product in the harvest liquor is sometimes as low as 1 mg/lbut usually is on the order of 100 mg/l. The larger proteins inparticular are very fragile, and their conformation is essential totheir biological function. Because of their complex structure andfragility, they must be treated with relatively low fluid shear, andpreferably with only minimal and short duration contact with surfaces.The presence of proteases in the process liquor often mandates thatpurification be conducted as quickly as possible.

The major performance measures of chromatography techniques areproductivity and peak resolution. Productivity refers to specificthroughput. It is a measure of the mass of solute that can be processedper unit time per unit volume of chromatography matrix. Generally,productivity improves with increases in 1) the surface area per unitvolume of the matrix, 2) the rate of solute mass transfer to the sorbentsurface, 3) the rate of adsorption and desorption, and 4) the fluid flowvelocity through the matrix.

Resolution is a measure of the degree of purification that a system canachieve. It is specified by the difference in affinity among solutes inthe mixture to be separated and by the system's inherent tendency towarddispersion (bandspreading). The former variable is controlled by thenature of solutes in the process liquor and the chemical properties ofthe interactive surface of the chromatography medium. Bandspreading iscontrolled primarily by the geometry of the chromatography matrix andthe mass transfer rates which obtain during the chromatographyprocedure. Resolution is improved as theoretical plate height decreases,or the number of plates increases. Plate height is an indirect measureof bandspreading relating to matrix geometric factors which influenceinequities of flow, diffusion, and sorption kinetics.

It obviously is desirable to maximize productivity and to minimizebandspreading in a matrix designed for preparative chromatography.However, the design of a chromatography matrix inherently ischaracterized by heretofore unavoidable constraints leading to tradeoffsamong objectives. For example, the requirement of a large surface areato volume ratio is critical to throughput, and practically speaking,requires the matrix to be microporous. Such microporous particulatematerials are characterized by a nominal pore size which is inverselyrelated to the surface area of the particles and a nominal particlediameter which dictates the pressure drop for a given packed column.Operations with rapid flows and small microporous particles require highoperating pressures and promote bandspreading. Increasing the size ofthe particles decreases back pressure. Increasing the size of the poresdecreases surface area and, together with increasing particle size,results in significant decreases in productivity. If rigid particles areused together with high pressures, gains in productivity can be achieved(e.g., HPLC), but plate height, the measure of bandspreading, isproportional to the flow rate of liquids through the matrix. Thus, whenhigh surface area porous particles are used, as fluid velocity isincreased, plate height increases and peak resolution decreases.

The phenomenon of bandspreading generally is described by the function:

    H=Au.sup.1/3 +B/u+Cu                                       (Eq.-1)

wherein A, B, and C are constants for a particular chromatographycolumn, u is the velocity of fluid through the bed, and H is the plateheight. The A term is a measure of bandspreading caused by longitudinaldiffusion, i.e., a term accounting for the fact that there is a slowmolecular diffusion along the axis of a column. The B term accounts forthe fact that a fluid passing through a column can take many differentpaths. This is often related to as "eddy diffusion". The A and B termsdominate bandspreading phenomena in a given matrix at low fluid flowvelocities. At high velocities, the contribution of these factors tobandspreading is minimal, and the phenomenon is dominated by the C term.This term accounts for stagnant mobile phase mass transfer, i.e., theslow rate of mass transfer into the pores of the particles of thematrix. As a solute front passes through a column at a given velocity,some solute will penetrate the pores and elute later than the front.

The degree of bandspreading traceable to the C term is related toparticle diameter, solute diffusion coefficient inside the pores, poresize, and the velocity of the solute outside the pores. Morespecifically, the C term is governed by the expression: ##EQU1## whereinc is a constant, d is the diameter of the particle, and D_(eff) is theeffective diffusion coefficient of the solute within the pore. Tomaximize throughput, fluid velocity should be high. But as is apparentfrom the foregoing expression, increasing velocity increases masstransfer limitations due to pore diffusion and therefore leads toincreased bandspreading and decreased dynamic loading capacity. Notealso that bandspreading increases as a function of the square of theparticle size. Thus, attempts to increase throughput at a given pressuredrop by using higher liquid flow rates among the intersticies of largeparticles produces geometric increases in bandspreading caused by slowintraparticle diffusion.

It is also apparent from equation 2 that bandspreading can be reduced byincreasing the effective diffusion constant. Of course, diffusion rateis an inverse function of the molecular weight of the solute and isdependent on concentration gradients. Thus, proteins having a highmolecular weight typically have diffusion constants in the range of 10⁻⁷to 10⁻⁸ cm² /sec. For this reason, chromatographic separation ofproteins can produce levels of bandspreading not encountered with lowermolecular weight solutes. Furthermore, the effective diffusivity throughthe pores of the particles is lower than the diffusivity in freesolution. This is because diffusion is hindered in pores having meandiameters comparable to the molecular diameter of the solute, e.g., nomore than about a factor of 10 or 20 greater than the solute. Effectivediffusivity differs from ideal also because the solute must diffuse intothe particle from fluid passing by the particle. Increasing convectiveflow in what is virtually a perpendicular direction to the direction ofdiffusion produces an effective diffusion rate somewhat lower than theideal.

Effective diffusivity also is decreased during loading of the surface ofthe sorbent with solute. This phenomenon has been explained as being dueto occlusion of the entrance of the pore by adsorbed protein. As proteinmolecules begin to diffuse into the porous matrix, they are thought tosorb at the first sites encountered, which typically lie about theentryway of the pore. It is often the case that the dimensions of amacromolecular solute are significant relative to the diameter of thepore. Accordingly, after a few molecules have been sorbed, the entranceto the pore begins to occlude, and the passage of solute into theinterior of the pore by diffusion is hindered. As a result of thisocclusion phenomenon, mass transfer of solute into the interior of thesorbent particle is reduced further.

Many of the negative effects on plate height caused by stagnant mobilephase loading in porous particles may be alleviated by decreasingparticle size, and therefore pore length. However, as noted above, thisstrategy requires operation at increased pressure drops.

Recently, it was suggested by F. E. Regnier that chromatographyparticles having relatively large pores may enhance performance byallowing faster diffusion of large molecules. It was thought thatincreasing pore size might alleviate the pore entry clogging problem andpermit diffusion into the particles relatively unhindered by poreeffects.

There is a different class of chromatography systems which are dominatedby convective processes. This type of system comprises sorbent surfacesdistributed along flow channels that run through some type of bed. Thebed may be composed of non-porous particles or may be embodied as amembrane system consisting of non-porous particle aggregates, fibermats, or solid sheets of materials defining fabricated holes. Thechannels of the non-porous particle systems are formed, as with thediffusion bound systems, by the interstitial space among the particles.The space between fibers forms channels in fiber mats. Channels formedby etching, laser beam cutting, or other high energy processes typicallyrun all the way through the membrane, whereas the former type ofchannels are more tortuous.

In these systems, solute is carried to the sorbent surface by convectiveflow. Solute may be transported for relatively long distances withoutcoming into contact with sorbent surface because channel dimensions areoften quite large (0.2 to 200 μm). The flow is generally laminar, andlift forces divert solutes away from channel walls. These drawbacks tomass transfer of solute to the solid phase can be serious and present asignificant obstacle to high flow rates. Thus, channels must be long toensure that solute will not be swept through the sorbent matrix whileescaping interactive contact. The provision of smaller diameter channelsincreases required operational pressure drops. If velocity is reduced,throughput obviously suffers. Still another disadvantage of theconvective transport system is that it inherently has a relatively lowsurface area and accordingly less capacity than other systems of thetype described above.

Elimination of the pores from a particulate sorbent can allowseparations to be achieved very rapidly. For example, 2 μm non-porousparticle columns can separate a mixture of seven proteins in less thanfifteen seconds. However, this approach cannot solve the engineeringchallenges presented by the requirements for purification of highmolecular weight materials as dramatically demonstrated in the table setforth below.

    ______________________________________                                        CHARACTERISTICS OF NON-POROUS                                                 PARTICLE COLUMNS                                                              ______________________________________                                        Particle 10    5.0    2.0  1.0   0.5  0.1    0.05                             Size (μm)                                                                  Surface  0.6   1.0    3.1  6.3   10   63     105                              Area (m.sup.2 /ml)                                                            Pressure 17    68     425  1700  6800 17000  68000                            Drop (psi/cm                                                                  of bed height)                                                                ______________________________________                                    

As illustrated by these data, small particles, whether present in packedcolumns or membranes, have very serious pressure problems at particlesizes sufficient to provide large surface areas and large loadingcapacity. In contrast, 300 Å pore diameter particles in the 5 to 100 μmrange have from 70 to 90 m² /ml of surface area, while a 1,000 Åmaterial has an area on the order of 40 to 60 m² /ml.

A chromatography cycle comprises four distinct phases: adsorption, wash,elution, and reequilibration. The rate limiting step in each stage isthe transport of molecules between the mobile fluid and the staticmatrix surface. Optimum efficiency is promoted by rapid, preferablyinstantaneous mass transfer and high fluid turnover. During sorbentloading, with a step concentration of the protein, fewer molecules aresorbed as the velocity of mobile phase in the bed increases. Theconsequence is that some protein will be lost in the effluent or willhave been lost as "breakthrough". If the breakthrough concentration islimited to, for example, 5% of the inlet concentration, that limit setsthe maximum bed velocity which the bed will tolerate. Furthermore,increases in bed velocity decrease loading per unit surface area.

As should be apparent from the foregoing analysis, constraintsconsidered to be fundamental have mandated tradeoffs among objectives inthe design of existing chromatography materials. Chromatography matrixgeometry which maximizes both productivity and resolution has eluded theart.

It is an object of this invention to provide the engineering principlesunderlying the design of improved chromatography materials, to providesuch materials, and to provide improved chromatography methods. Anotherobject is to provide chromatography particles and matrices,derivatizable as desired, for the practice of a new mode ofchromatographic separation, named herein perfusion chromatography,characterized by the achievement a high fluid flow rates but manageablepressure drops of extraordinarily high productivities and excellent peakresolution. Another object is to provide improved methods of separatingand purifying high molecular weight products of interest from complexmixtures. Another object is to overcome the deficiencies of bothconvection bound and diffusion bound chromatography systems. Stillanother object is to provide a chromatography procedure and matrixgeometry wherein effective plate height is substantially constant over asignificant range of high fluid flow velocities, and at still highervelocities increases only modestly.

These and other objects and features of the invention will be apparentfrom the drawing, description, and claims which follow.

SUMMARY OF THE INVENTION

It has now been discovered that chromatography matrix geometries can bedevised which, when exploited for chromatographic separations above athreshold fluid velocity, operate via a hybrid mass transport system,herein called perfusion, which couples convective and diffusive masstransport. The matrix materials are extraordinary in that they permitorder of magnitude increases in productivity without significantlycompromising resolution. Furthermore, surprisingly, the most dramaticimprovements are achieved with relatively large particles which permitproductive operation at relatively low column pressure drops. Perfusionchromatography uncouples bandspreading from fluid velocity, succeeds inachieving unprecedented combinations of throughput and resolution, anduncouples that which determines pressure drop from that which determinesmass transport.

Perfusion chromatography may be used for rapid analysis and also inpreparative contexts. Perhaps its optimum use is in separation andpurification of large biologically active molecules such aspolypeptides, proteins, polysaccharides, and the like. The technique hasless advantage for small molecules with their much higher diffusionconstants and inherently faster mass transport. However, even with lowmolecular weight materials such as sugars and alcohols, perfusionchromatography can be exploited to advantage, particularly when usinglarge particles as a chromatography matrix material where the distanceover which diffusion must act is relatively large. A use for perfusionchromatography is in separation of solutes comprising biologicalmolecules, e.g., molecules of biological origin or having biologicalactivity.

A key to achieving these goals is the availability of matrix materialsdefining at least primary and secondary sets of pores, i.e., "first" and"second" sets of interconnected pores, with the members of the firstpore set having a greater mean diameter than the members of the secondpore set. The matrix also defines surface regions which reversiblyinteract with the solutes to be separated and which are disposed influid communication with the members of the second pore set. Thedimensions of the first and second pore sets are controlled such thatwhen a mixture of solutes is passed through the matrix above a thresholdvelocity, convective flow is induced through both pore sets. The domainof perfusion chromatography begins when the rate of fluid flow increasesto a level where convective flow through the members of the second poreset exceeds the rate of diffusion of the solute through those pores. Atthe outset, the advantages over conventional chromatography techniquesare modest, but as superficial bed velocities increase, dramaticincreases in productivity are achieved.

The mean diameter of the members within each of the first and secondpore sets can vary significantly. In fact, one preferred matrix materialcomprises a second pore set having a plurality of interconnected poresubsets which permit convective flow, and smaller subpores comprisinglooping pores or blind pores communicating with pores where convectionoccurs. The subpores contribute most significantly to the surface areaof the matrix. Most solute/matrix interactions occur in these subpores.Mass transfer between the surface and the members of the interconnectedpore subsets occurs by way of diffusion. This type of geometry producesa second pore set with a wide distribution of mean pore diameters. Inanother embodiment, one or both of the first and second pore setscomprise pores having a narrow distribution of pore diameters such thatthe diameter of 90% of the pores in the set falls within 10% of the meandiameter of all of the pores in the set. In a preferred embodiment thesubpores have a mean diameter less than about 700 Å. Preferably, thefluid mixture of solutes to be separated is passed through the matrix ata rate such that the time for solute to diffuse to and from a surfaceregion from within one of the members of the second pore set is nogreater than about ten times the time for solute to flow convectivelypast the region.

This type of matrix geometry has several advantages. First, in a matrixof sufficient depth, all of the liquid will pass through the second poreset numerous times, although the pressure drop is determined primarilyby the larger mean diameter of the first pore set. Second, in thepreferred packed particle matrix embodiment, with respect tointraparticle stagnant mobile phase constraints, the perfusive matrixbehaves like a matrix of packed, non-porous particles, or porousparticles of very small diameter, yet pressure and velocity requirementsare characteristic of much larger particle beds. Third, mass transportbetween the sorbent surface and mobile phase is effected primarily byconvective flow. Diffusion still must occur, but the diffusion paths areso much shorter that this constraint becomes mimimal.

In the chromatography process of the invention, the fluid mixtures,eluents, etc. preferably are passed through the matrix at a bed velocitygreater than 1000 cm/hr, and preferably greater than 1500 cm/hr.Productivities exceeding 1.0 and often 2.0 mg total protein sorbed perml of sorbent matrix per minute are routinely achieved. In the preferredpacked particle matrices, the particles preferably have a mean diameterof at least about 8.0 μm, and preferably greater than 20 μm. Since, as arule of thumb, the mean diameter of the pores defined by theintersticies among roughly spherical particles is approximatelyone-third the particle diameter, these interstitial pores, comprisingthe first pore set, will have a mean diameter on the order of about 3.0μm, and for the larger particles, 7-20 μm or larger. The second pore setin this embodiment consists of the throughpores within the particles.Effective perfusive chromatography requires the ratio of the meandiameter of the particles to the mean diameter of the second pore setsto be less than 70, preferably less than 50. The dimensions of the firstand second pore sets preferably are such that, at practical flowvelocities through the bed, the ratio of the convective flow velocitiesthrough the first pore set, i.e., the intersticies among the particles,to the second pore set, i.e., the throughpores in the particles, iswithin the range of 10 to 100.

The chromatography matrices of the invention may take various formsincluding beds of packed particles, membrane-like structures, andfabricated microstructures specifically designed to embody theengineering principles disclosed herein. However, a preferred form is apacked bed of particles having a mean diameter greater than 10 μm, eachof which define a plurality of throughpores having a mean diametergreater than about 2,000 Å. The particles comprise rigid solids whichpresent a large interior solute-interactive surface area in direct fluidcommunication with the throughpores. Currently preferred particlescomprise a plurality of interadhered polymeric spheres, herein termed"porons", which together define interstitial spaces comprising thesubpores and throughpores. The subpores preferably have an averagediameter in the range of 300 Å to 700Å. This approach to the fabricationof chromatography particles and matrices of the invention also permitsthe manufacture of particles defining branching pores, communicatingbetween the throughpores and the subpores, which have intermediate meandiameters. Preferably, the throughpores, subpores, and anyinterconnecting pores are anisotropic.

In this particle fabrication technique, it is preferred to build theparticles from porons to produce small poron clusters, and then toaggregate the clusters, and then possibly to agglomerate the aggregatesto form particles of macroscopic size, e.g., greater than 40 μm, whichoptionally may themselves be interadhered to produce a one-piece matrix.This approach results in production of a second pore set comprising aplurality of throughpore subsets and subpores of differing meandiameters. Preferably, the ratio of the mean diameter of any consecutivesubset of throughpores is less than 10. The ratio of the mean diameterof the smallest subset of throughpores to the mean diameter of thesubpores Preferably is less than 20. The ratio of the mean diameter ofthe first pore set, here defined by the intersticies among theinteradhered or packed particles, and the largest subset ofthroughpores, Preferably is less than 70, more preferably less than 50.

These and other objects and features of the inventions will be apparentfrom the drawing, description, and claims which follow.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1A, 1B, 1C, 1D, and FIG. 2 are schematic representations ofparticle/matrix geometries useful in explaining perfusionchromatography;

FIG. 3 is a graph of productivity versus fluid velocity (V_(Bed)) andoperational pressures (ΔP) illustrating the domains of diffusivelybound, convectively bound, and perfusive chromatography systems;

FIG. 4A, 4B, and 4C are scanning electron micrographs of a macroporouschromatography particle useful for fabricating matrices for the practiceof perfusion chromatography: 4A is 10,000×; 4B is 20,000×, and 4C is50,000×;

FIG. 4D is a schematic diagram illustrating the fluid dynamics which arebelieved to be controlling during perfusion chromatography using theparticle structure shown in FIG. 4A-4C;

FIG. 5A is a schematic cross-section of a chromatography column;

FIG. 5B is a schematic detail of the circle B shown in FIG. 5A;

FIG. 5C is a schematic diagram illustrating one idealized structure fora perfusion chromatography matrix element;

FIG. 6 is a solute breakthrough curve of outlet concentration/inletconcentration vs process volume in milliliters illustrative of thedifferences in kinetic bahavior between conventional and perfusionchromatography;

FIG. 7 is a bar graph of capacity in mgs for a bed of a given volume vs.superficial fluid flow velocity through the bed comparing the adsorptioncapacity of a typical perfusive column with a conventional diffusivecolumn;

FIG. 8 is a graph of bed velocity in cm/hr vs. throughpore size inangstroms showing the maximum and minimum pore sizes able to achieve aPeclet number greater than 10 at a given diffusion coefficient andparticle size;

FIG. 9 is a graph of minimum pore mean diameter in angstroms vs.particles diameter in μm illustrating the perfusive domain at variousVbed given the assumptions disclosed herein; and

FIGS. 10 through 22 are graphs presenting various data demonstrating theproperties of perfusion chromatography systems.

Like reference characters in the respective drawn figures indicatecorresponding parts.

DESCRIPTION

In this specification the nature and theoretical underpinnings of therequired matrix structures and operational parameters of perfusionchromatography will first be disclosed, followed by engineeringprinciples useful in optimization and adaptation of the chromatographyprocess to specific instances, disclosure of specific materials that areuseful in the practice of perfusion chromatography, and examples ofperfusion chromatography procedure using currently available materials.

Broadly, in accordance with the invention, perfusion chromatography ispracticed by passing fluids at velocities above a threshold levelthrough a specially designed matrix characterized by a geometry which isbimodal or multimodal with respect to its porosity. Perhaps the mostfundamental observation relevant to the new procedure is that it ispossible to avoid both the loss of capacity characteristic of convectionbound systems and the high plate height and bandspreadingcharacteristics of diffusion bound systems. This can be accomplished byforcing chromatography fluids through a matrix having a set of largerpores, such as are defined by the intersticies among a bed of particles,and which determine pressure drops and fluid flow velocities through thebed, and a set of pores of smaller diameter, e.g., anisotropicthroughpores. The smaller pores permeate the individual particles andserve to deliver chromatography fluids by convection to surface regionswithin the particle interactive with the solutes in the chromatographyfluid.

The relative dimensions of the first and second pore sets must be suchthat, at reasonably attainable fluid velocities through the bed,convective flow occurs not only in the larger pores but also in thesmaller ones. Since fluid velocity through a pore at a given pressure isa function of the square of the pore radius, it can be appreciated thatat practical fluid velocities, e.g., in the range of 400 to 4,000cm/hr., the mean diameter of the two sets of pores must be fairly close.As a rule of thumb, the mean diameter of pores defined by theintersticies among spherical particles is about one third the diameterof the particles. Thus, for example, particles having a mean diameter of10 μm and an average throughpore diameter of 1,000 Å, when close packedto form a chromatography bed, define first and second sets of poreshaving mean diameters of approximately 3 to 4 μm and 0.1 μm,respectively. Thus, the mean diameter of the larger pores is on theorder of thirty to forty times that of the smaller pores. Under thesecircumstances, very high pressure drops are required before anysignificant fraction of the fluid passes by convection through thesmaller pores within the particles.

Experiments with this type of material have failed to indicate perfusiveenhancement to mass transport kinetics. Thus, at the flow rates tested,mass transport into the 10 μm particles appear to be dominated bydiffusion. Stated differently, any convective flow within thethroughpores does not contribute significantly to the rate of masstransport. Obviously, more conventional solid chromatography media suchas most silica based materials, agars, dextrans and the like, which havemuch smaller pores (generally between approximately 50 and 300Å) andlarger mean particle sizes (20 μm to 100 μm), cannot be operatedpractically in the perfusive mode. There simply is no realistic flowvelocity attainable in a practical system which results in anysignificant convective flow within their secondary micropores.Generally, larger mean diameter throughpores, or more specifically, asmaller mean diameter ratio between the first and second pore sets, isrequired to practice perfusion chromatography.

The nature of perfusion chromatography and its required matrix geometrymay be understood better by reference to FIGS. 1A through 1D. These areschematic diagrams roughly modeling the fluid flow in various types ofchromatography matrices showing in schematic cross section one region ofthe matrix. The chromatography particle or region is accessed by a majorchannel 10 on the "north" side which leaves from the "south" side andmay or may not have a circumventing channel which allows the fluidmobile phase containing dissolved solutes to by-pass the particle. Theparticles themselves comprise a plurality of solute interactive surfaceregions represented by dots which must be accessed by solute molecules.The nature of these regions depends on the chemistry of the activesurface. The process of this invention is independent of the nature ofthe active regions which, in various specific embodiments, may take theform of surfaces suitable for cationic or anionic exchange,hydrophobic/hydrophilic interaction, chelation, affinity chromatography,hydrogen bonding, etc. Low plate height and minimization ofbandspreading require rapid mass transfer between the interactivesurface regions and the fluid mobile phase. High capacity requires bothrapid mass transfer and the presence of a large number of interactiveregions, i.e., high surface area. Solute is transported by twomechanisms: convection, which is determined by pore size, pressure drop,pore length and tortuosity and local geometry about the entry and exitof the pore; and intrapore diffusion, which is a function primarily ofthe molecular dimensions of the various solutes, the dimensions of thepore, and of concentration gradients.

The mechanism of solute interaction with the matrix in two types ofconvection bound chromatography systems will be disclosed with referenceto FIGS. 1A and 1B; diffusive bound systems with reference to FIG. 1C;and perfusive systems with reference to FIG. 1D.

FIG. 1A represents the chromatography matrix comprising close packednon-porous particles. The interior of the particles is barred to accessby solute molecules. The only interactive surface elements that areavailable to the solute molecules are those arrayed about the exteriorsurface of the particle. FIG. 1B represents a membrane-likechromatography "particle" (actually a region in a solid matrix) havingthroughpores and interactive surface regions disposed along the walls.The geometry of FIG. 1B is analogous to filter beds and polymer webmorphologies (e.g., paper and membrane filters) and to bundles ofnon-porous fibers or tubes. In the morphologies of FIGS. 1A and 1B, onlythe outside surface of the chromatography mandrel contributes to thecapacity of the matrix. The surface area to volume ratio of thesegeometries is relatively low, and they are therefore inherently lowproductivity systems. Provided the flow paths 10 are long enough, veryrapid separations and high resolution without breakthrough can beachieved because the distance a solute molecule must diffuse from aconvective channel to an interactive surface element is small. Ofcourse, an attempt to increase the number of interactive surfaceelements (surface area) by decreasing particle size (FIG. 1A) ordecreasing pore diameter (FIG. 1B) amounts to a tradeoff for higheroperating pressure. Increasing the fluid velocity through the bed beyondthe optimal degrades performance.

In FIG. 1C, the interactive surface elements are disposed about theinterior of the particle and, per unit volume of particle, are far morenumerous. Here, the interior of the matrix is accessible via small pores12. Solute can pass through these pores only by diffusion, or by acombination of diffusion coupled with an extremely slow convection whichhas no significant effect on the overall kinetics of mass transport.Accordingly, solute molecules are moved from flow channel 10 into theinterior of the particle by slow diffusive processes. This constraintcan be alleviated by making the particles smaller and thereforedecreasing the distance required to be traversed by diffusion. However,again, this is achieved at the expense of greatly increasing requiredoperational pressure drops. For macromolecules such as proteins, theeffective diffusivity within the pores is decreased further by the poresurface hindrance and occlusive effects as discussed above.

When such porous particles are fully loaded, i.e., solute molecules havediffused along the pores and are now occupying all interactive surfaceregions, the matrix is washed, and then elution commences. These suddenchanges in conditions induce solutes to evacuate the particles. This,again, is accomplished by slow diffusion. Gradually, solute from thecenter of the particle arrives at the ring channel to be carried off byconvection. This delay in "emptying" the particle by diffusion is acontributing cause of the trailing tail on a chromatography pulse whichreduces resolution. The rate at which the particle can be loaded andunloaded determines the kinetics of the chromatography process. Clearly,the faster solute can escape, the shorter the time for all of the soluteto arrive at the chromatography column's output, and hence the shorterthe straggling tail and the less bandspreading. Increasing fluidvelocity in channels 10 above an optimal level has no positive effect onthroughput and causes plate height to increase and resolution todecrease.

FIG. 1D models a matrix particle suitable for perfusion chromatography.As illustrated, in addition to channels 10 having a relatively largemean diameter (defined by the intersticies among particles in theparticulate matrix embodiment) the matrix also comprises a second set ofpores 14, here embodied as throughpores defined by the body of theparticle. The mean diameter of the pores 14 is much larger than thediffusive transport pores 12 of the conventional chromatographypartricle depicted in FIG. 1C. The ratio of the mean diameters of pores10 and 14 is such that there exists a fluid velocity threshold which canpractically be achieved in a chromatography system and which induces aconvective flow within pores 14 faster than the diffusion rate throughpores 14. Precisely where this threshold of perfusion occurs depends onmany factors, but is primarily dependent on the ratio of the meandiameters of the first and second pore sets, here pores 10 and 14,respectively. The larger that ratio, the higher the velocity threshold.

Actually, the bed velocity corresponding to the threshold is that atwhich intraparticle convection begins to influence transport kinetics.At much higher velocities convection dominates and significantperformance improvements are observed.

In matrices comprising close packed 10 μm particles, tne mean diameterof pores 10 (comprising the intersticies among the particles) is on theorder of 3 μm. Such 10 μm particles having throughpores of about 1,000 Åin diameter (0.1 μm) do not perfuse at practical flow rates; 10 μmparticles having a plurality of pores within the range of 2000 Å to10,000 Å (0.2 mm-1.0 μm) perfuse well within a range of high fluidvelocities through the bed (approx. 1000 cm/hr or greater). In matricescomprising closepacked particles having 1,000 Å mean diameterthroughpores, the ratio of the mean diameter of the first to the secondpore set is about 3.3/0.1 or approximately 33. For the corresponding4,000Å mean diameter throughpore particle, the ratio is approximately8.3. While these numbers are rough and are dependent on manyassumptions, the ratio of the mean diameters of the first and secondpore sets effective to permit exploitation of the perfusionchromatography domain with operationally practical flow rates isbelieved to lie somewhere within this range, i.e., 8-33.

Again referring to FIG. 1D, it should be noted that mass transport toregions within the particle and into the vicinity of the interactivesurface elements is dominated by convection. While diffusive masstransport is still required to move solutes to and from pores 14 and theinteractive surface regions, the distance over which diffusive transportmust occur is very significantly diminished. Thus, with respect tobandspreading and mass transfer kinetics, the bed behaves as if it werecomprised of very fine particles of a diameter equal approximately tothe mean distance between adjacent throughpores (e.g., on the order of1.0 μm with currently available materials). It has a high surface areato volume ratio and rapid kinetics. However, operating pressure dropessentially is uncoupled from these properties as it is determined bythe larger dimensions of channels 10 comprising the first pore set.

At low velocities through the matrix, perfusive particles such as theparticles schematically depicted in FIG. 1D behave similarly todiffusion bound conventional chromatography materials. At lowvelocities, convective flow essentially is limited to the larger firstset of pores 10. Convective flow within pores 14 is so small as to benegligible, transport from within the particle to the flow channels 10takes place through diffusion. The larger pores permit more optimaldiffusion rates as occlusive effects and diffusion hindrance withinpores are somewhat alleviated.

As the fluid velocity in the bed (and pressure drop) is increased, therecomes a point when the convective flow rate through the pores 14 exceedsthe rate of diffusion and operation in the perfusive mode commences.This flow rate is about 300 cm/hr for 10 μm chromatography particleshaving 4,000 Å pores for a solute having a pore diffusivity of 10⁻⁷ cm²/sec. Above this threshold, it will be found that increased pressuredrop and velocity permit increased throughput per unit volume of matrixnever before achieved in chromatography systems. At about 600 cm/hrproductivities approximately equal to the highest heretofore achievedare observed. At 1000 cm/hr to 4000 cm/hr, extraordinary productivitiesare achieved. Furthermore, these productivities are achieved without theexpected increase in bandspreading, i.e., decrease in resolution.

While this behavior seemingly violates long established physicalprinciples governing the general behavior of chromatography systems,recall that at high velocities the primary contributor to bandspreadingis stagnant mobile phase mass transfer within the particle, or the "Cterm" discussed above. Thus, in the perfusive system: ##EQU2##

However, D_(Eff) which, at low fluid velocities through the matrix, is ameasure of the effective diffusion of solute into the pores 14 and intocontact with the surface regions, becomes, in the perfusive mode, aconvection dominated term. In general, one can approximate D_(Eff) asthe sum of a diffusive element (pore diffusivity) and a convectiveelement (pore velocity x particle diameter). Calculated in this wayD_(Eff) is a conservative estimate which ignores the different drivingforces for the two modes of transport. For any given fluid velocity andbed geometry operated in the perfusive mode, the ratio of fluid velocitywithin the second pore set to superficial fluid velocity in the bed willbe given by: ##EQU3## wherein α is a constant. Thus, fluid velocitywithin the members of the second pore set becomes α V_(bed), and theplate height due to the C term effectively becomes: Since u representsthe velocity of fluid in the bed, the plate height reduces to:

    H=c'd                                                      (Eq. 5)

Thus, the C term becomes substantially independent of bed velocity inthe perfusion mode. It will not be completely independent because, asnoted above, diffusion still will play a part in mass transfer betweenconvective channels and sorptive surface regions. At some high V_(Bed),the system will once again become kinetically bound by mass transferresistance due to diffusion into subpores.

One measure of the mass transfer of a solute through a pore is given bya characteristic Peclet number (P_(e)), a dimensionless quantity equalto VL/D, where V is the convective velocity through the pore, L is itslength, and D is the diffusivity of the solute through the pore. In theprior art systems, under all regimes, the Peclet number which describesthe ratio of convective to diffusive transport within the pores of achromatography material was always much less than one. In perfusivechromatography, the Peclet number in the second set of pores is alwaysgreater than one.

Referring to FIG. 2, a conceptual model of a region of matrix 5 depictedin cross-section has three types of pores; the members of the first poreset 10; throughpores 14 comprising the members of the second pore set;and subpores 16. These, respectively, are characterized by Pecletnumbers P_(e) I, P_(e) II, and P_(e) III, given below: ##EQU4## whereinEpsilon is the void volume of the bed, d_(p) is the diameter of theparticle (representitive channel length average over a particle,includes a correction for tortuosity), L_(d) is the depth of the subpore, D_(EFF) is the effective diffusivity within the throughpore, D₁ isthe restricted diffusivity in the throughpores, and D₂ is the restricteddiffusivity in the subpores.

The kinetics of chromatography in general is adversely affected by highP_(e) I, low P_(e) II, and high P_(e) III. Thus, chromatographicperformance is enhanced if effective diffusivity increases, or ifparticle size decreases or V_(Bed) decreases. At high P_(e) I, highconvection rates sweep the solute past the throughpores, thusdiscouraging mass transfer. On the other hand, in the second pore set ahigh Peclet number is preferred. When P_(e) II is high, mass transferincreases as the convective velocity takes over from diffusion as thedominant mechanism in mass transport through a particle. Within subpore16, a low Peclet number is desired. When P_(e) III is low, diffusion tothe active surface within the sub pores is faster than flow through theparticle, and consequently dynamic capacity remains high.

Increasing mobile phase velocity deteriorates the performance ofdiffusive systems, but has far less effect with perfusion. Instead, anincrease in bed velocity yields a corresponding increase in porevelocity which controls the mass transfer kinetics inside the support.Thus, with the correct geometric relationship of the matrix, proper flowrates, pressure drops, and fluid viscosities, a domain is obtained wherethe mass transport characteristics of the system favor simultaneouslyvery high throughput and high resolution separations.

FIG. 3 is a graph of productivity in milligrams of solute per second perml of matrix versus bed velocity and pressure drops. The graphillustrates the difference in behaviors among diffusively boundchromatography systems, convectively bound systems, and perfusivesystems. As shown, in conventional diffusion limited system as velocityand pressure are increased productivity increases until a maximum isreached, and further increases in V_(Bed) result in losses inproductivity, typically resulting in breakthrough or loss in dynamicloading capacity well prior to a bed velocity of about 400 cm/hr. Inconvectively bound systems, much higher fluid velocities and pressuredrops may be used. For a bed of sufficient length, productivity willincrease steadily, possibly up to as high as 4,000 cm/hr fluid flowrate, but the gains in productivity are modest due to the inherently lowsurface area and binding capacity. For perfusion systems, increases inbed velocity at the outset increase productivity in a manner similar todiffusively bound systems. However, above a threshold bed velocity, whenthe Peclet number in the throughpores becomes greater than 1, orconvective flow velocity exceeds diffusive flow velocity within thepores, the perfusive realm is entered. Further increases in velocityserve to increase convection within the pores and increase masstransport. At some high flow rate, the perfusive system becomesdiffusively bound because the time it takes for a solute molecule todiffuse to and from a throughpore to an interactive surface regionbecomes much greater than the time it takes a solute molecule to move byconvection on past the region. However, the distance over whichdiffusion must act as the transport mechanism is much smaller than inconventional diffusion bound systems. Thus, optimal perfusiveperformance continues at least through the bed velocity where thesubpore diffusion time is ten times as great as the throughporeconvective time.

To evaluate the implications of perfusive kinetics on chromatography bedsorption, existing models were modified and used to simulate thesorption process. Column sorption behavior often is shown in the form ofsolute "breakthrough" curves which comprise a plot of effluentconcentration vs. time. For a given column, if the flow rate of the feedto the sorptive surface is sufficiently slow to permit the contact timebetween the solute and the sorbent to be long enough to overcome finitemass transfer rates, equilibrium sorption is achieved. In this case, theinitial amount of solute loaded onto the column is sorbed and no soluteappears in the column effluent. When sufficient solute is loaded ontothe column to saturate the sorbent phase, no more solute can be sorbedand the solute concentration in the effluent matches that of the feed.In practice, in diffusively bound systems, sorption deviates from theequilibrium limit due to slow mass transport rates.

FIG. 6 is a graph of breakthrough (outlet concentration/inletconcentration) vs. processed volume illustrating a fundamentaldifference between conventional diffusive bound and perfusivechromatography systems. The curves were calculated assuming a feedprotein concentration of 5 mg/ml, 3.25 mls of sorbent, a column 5.4 cmlong and 1.1 cm wide, a column void fraction of 0.35, an availablesurface area of 40 mg/ml of matrix, and a sorption constant of 1 ml/mg.As illustrated in FIG. 6, in conventional chromatography procedures,increasing the bed velocity has the effect of skewing the curve from theideal. At 100 cm/hr, the breakthrough curve is almost completelyvertical because solute/sorbent equilibrium is established. As linearbed velocity increases, mass transfer rates begin to dominate andpremature solute breakthrough occurs. Compare, for example, the curvesfor 500, 1,000, and 2000 cm/hr. At very high bed velocities, e.g., 5,000cm/hr, premature solute breakthrough is severe, because a fraction ofthe feed solute passes through the column without being sorbed, as shownby the immediate jump in effluent solute concentration.

In contrast, for a similar column having the same simulation conditionwherein the matrix is perfusive, the predictive solute breakthroughcurve is much sharper and is similar to the equilibrium sorption limit.This predicted behavior was verified by experiment, as is discussedbelow.

In preparative chromatography, frontal column loading typically isterminated at the point where solute effluent concentration reaches 10%of the feed concentration. The amount of feed processed until that pointdefines the column capacity. This capacity term is an importantdeterminant to overall productivity in the system, and typicallydecreases as the bed velocity increases in a diffusive particle column.Thus, at high bed velocities, for example, in excess of 2500 cm/hr, theinitial solute breakthrough is in excess of 10% of the feed, and thuscolumn capacity is effectively zero. In contrast, as shown in FIG. 7,the capacity of a perfusive particle column remains substantiallyconstant over a significant range of flow rates, since sorption kineticsare fast, and consequently, premature solute breakthrough occurs only atmuch higher levels.

PERFUSIVE MATRIX ENGINEERING

From the foregoing description many of the basic engineering goals to bepursued in the fabrication of matrix materials suitable for the practiceof perfusion chromatography will be apparent to those skilled in theart. Thus, what is needed to practice perfusion chromatography is amatrix which will not crush under pressure having a bimodal orpreferably multimodal pore structure and as large a surface area perunit volume as possible. The first and second pore sets which give thematerial its bimodal flow properties must have mean diameters relativeto each other so as to permit convective flow through both sets of poresat high V_(beds). The provision of subpores in the matrix is notrequired to conduct perfusion chromatography but is preferred because ofthe inherent increase in surface area per unit volume of matrix materialsuch a construction provides.

The matrix can take the form of a porous, one-piece solid of variousaspect ratios (height to cross-sectional area). Cross-sectional areasmay be varied from a few millimeters to several decimeters; matrix depthcan vary similarly, although for high fluid flow rates, a depth of atleast 5 mm is recommended to prevent premature breakthrough and what isknown as the "split peak" phenomenon. The structure of the matrix maycomprise a rigid, inert material which subsequently is derivatized toprovide the interactive surface regions using chemistries known to thoseskilled in the art. Alternatively, the structure may be made of anorganic or inorganic material which itself has a suitable soluteinteractive surface. Methods of fabricating suitable matrices includethe construction of particles which are simply packed into a column.These optionally may be treated in ways known in the art to provide abond between adjacent particles in contact. Suitable matrices also maybe fabricated by producing fiber mats containing porous particles whichprovide the chromatography surface. These may be stacked or otherwisearranged as desired such that the intersticies among the fibers comprisethe first pore set and the throughpores in the particles the second poreset. Matrices also may be fabricated using laser drilling techniques,solvent leaching, phase inversion, and the like to produce, for example,a multiplicity of anisotropic, fine pores and larger pores in, forexample, sheet-like materials or particulates which are stacked oraggregated together to produce a chromatography bed.

The currently preferred method for fabricating the matrices of theinvention involves the buildup of particles preferably having a diameterwithin the range of 5 μm to 100 μm from much smaller "building block"particles, herein referred to as "porons", produced using conventionalsuspension, emulsion, or hybrid polymerization techniques. Preferably,after fabrication of the particles, the interactive surface regions arecreated by treating the high surface area particles with chemistries toimpart, for example, a hydrophilic surface having covalently attachedreactive groups suitable for attachment of immunoglobulins for affinitychromatography, anionic groups such as sulfonates or carboxyl groups,cationic groups such as amines or imines, quaternary ammonium salts andthe like, various hydrocarbons, and other moities known to be useful inconventional chromatography media.

Methods are known for producing particles of a given size and givenporosity from porons ranging in diameter from 10 nm to 1.0 μm. Theparticles are fabricated from polymers such as, for example, styrenecross-linked with divinylbenzene, or various related copolymersincluding such materials as p-bromostyrene, p-styryldiphenylphosphine,p-amino sytrene, vinyl chlorides, and various acrylates andmethacrylates, preferably designed to be heavily cross-linked andderivatizable, e.g., copolymerized with a glycidyl moiety orethylenedimethacrylate.

Generally, many of the techniques developed for production of syntheticcatalytic material may be adapted for use in making perfusionchromatography matrix particles. For procedures in the construction ofparticles having a selected mean diameter and a selected porosity see,for example, Pore Structure of Macroreticular Ion Exchange Resins,Kunin, Rohm and Haas Co.; Kun et al, the Pore Structure ofMacroreticular Ion Exchange Resins; J. Polymer Sci. Part C, No. 16, pgs.1457-1469 (1967); Macroreticular Resins III: Formation of MacroreticularStyrene-Divinylbenzene Copolymers, J. Polymer Sci., Part A1, Vol. 6,Pgs. 2689-2701 (1968); and U.S. Pat. No. 4,186,120 to Ugelstad, issuedJan. 29, 1980. These, and other technologies known to those skilled inthe art, disclose the conditions of emulsion and suspensionpolymerization, or the hybrid technique disclosed in a Ugelstad patent,which permit the production of substantially spherical porons bypolymerization. These uniform particles, of a predetermined size on theorder of a few to a few hundred angstroms in diameter, are interadheredto produce a composite larger particle of desired average dimensioncomprising a large number of anisotropic throughpores, blindpores, andvarious smaller throughpores well suited for the practice of perfusionchromatography. The difference between the chromatography particlesheretofore produced using these prior art techniques and particlesuseful in the practice of this invention lies in the size of thethroughpores required for perfusion chromatography.

One source of particles suitable for the practice for perfusionchromatography is POLYMER LABORATORIES (PL) of Shropshire, England. PLsells a line of chromatography media comprising porons of polystyrenecross-linked with divinylbenzene which are agglomerated randomly duringpolymerization to form the particles. PL produced and subsequentlymarketed two "macroporous" chromatography media comprising particleshaving an average diameter of 8 μm to 10 μm and a particle-mean porediameter of 1000 Å and 4,000 Å. Actually, the mean pore diameter of theparticles represents an average between throughpores and subpores andthus bears little significance to the perfusion properties of thesematerials. The inventors named herein discovered that these particleshave mean throughpore diameters exceeding 2000 Å in the case of the"1000 Å" particle, and 6000 Å in the case of the "4000 Å" particle.These types of particle geometries can be made to perfuse underappropriate high flow rate conditions disclosed herein.

One type of PL particle, said to be useful for reverse phasechromatography, is an untreated polystyrene divinylbenzene (PSDVB). Itsinteractive surfaces are hydrophobic polymer surfaces which interactwith the hydrophobic patches on proteins. A second type of particle hasinteractive surface elements derivatized with polyethyleneimine and actas a cationic surface useful for anionic exchange. Both types ofparticles were produced in an ongoing effort initiated by F. E. Regnierto increase intraparticle diffusion of large solutes such as proteins byincreasing pore size. These particles were used by the inventors namedherein in the initial discoveries of the perfusive chromatographydomain.

These materials are sold under the tradenames PL-SAX 4000 for thepolyethyleneimine derivatized material and PLRP-S 4000 for theunderivatized material. While they are by no means optimal for perfusionchromatography, the pores defined by the intraparticle space in a packedbed of these materials and the throughpores in the particles have anappropriate ratio for achieving perfusion chromatography under practicalflow conditions.

Referring to FIGS. 4A, 4B, and 4C scanning electron micrographs of PL's10 μ, 4,000 Å porous particle are shown. As illustrated in FIG. 4C, thematerial comprises a multiplicity of interadhered porons, approximately1500 Å-2000 Å in diameter, which appear to be agglomerated at random toproduce an irregular high surface area, and a plurality of throughporesand subpores.

As shown in FIG. 4D, at a suitable V_(bed), chromatography fluids moveby convection through tortuous paths within the particle. The perfusivepores are anisotropic, branch at random, vary in diameter at any givenpoint, and lead to a large number of blind pores in which mass transportis dominated by diffusion. The blindpores and looping pores (subpores)generally have a mean diameter considerably smaller than the diameter ofthe porons (on the order of 1/3), and a depth which can vary from aslittle as a fraction of the diameter of the porons to 5 to 10 times thediameter of the porons.

FIGS. 5A and 5B illustrate scale factors of the geometry schematically.FIG. 5A is a cross section of a chromatography column showing amultiplicity of particle 20 each of which contacts its neighbors anddefine intersticies 22 which, in this form of matrix embodying theinvention, comprise the first pore set. As illustrated, the particlesare approximately 10 micrometers in diameter. The mean diameter of theintersticies vary widely but generally will be on the order of 1/3 ofthe mean diameter of the particles 20. Circle B in FIG. 5A is explodedtenfold in FIG. 5B. Here, the microstructure of the bed on a scale ofapproximately 1 micrometer is illustrated. The particles compriseclusters of porons illustrated as blank circles 24. The intersticiesamong the poron clusters define throughpores 14. The individual poronsmaking up clusters 24 here are illustrated by dots. At the next level ofdetail, i.e., 0.1 μm, or 1000 Å (not shown), a poron cluster 24 would beseen to comprise a roughly spherical aggregation of porons. In such astructure, the intersticies among the porons making up the aggregates 24are analagous to the diffusively bound particle of conventionalchromatography media such as is schematically depicted in FIG. 1C. Onlyin these would mass transport be diffusion dependent.

It may be appreciated that the chromatography matrices of the typedescribed above made from aggregations of smaller particles exhibit aself similarity over several geometric length scale and are thus"fractals" in the nomenclature of Mandlebrot.

The ideal perfusive chromatography matrix for preparative separation ofa given protein would comprise subpores dimensioned to permit diffusivetransport. Thus, the intersticies among the porons should be larger forhigher molecular weight proteins. This requires that larger porons beagglomerated. Fortunately, known polymerization techniques exploitingmicelle, emulsion, suspension, and "swollen emulsion" polymerization,and various techniques involving homogenization of immiscible mixturesare known. These techniques enable preparation of variously sizedparticles, as disclosed, for example in the references noted above andin Uniform Latex Particle, (Bangs, L. B., Seradyn, Inc, 1987). Thesemethods can be used to make particles of uniform mean diameter rangingfrom 200 Å up to about 20 μm. For the PL 1,000 and 4,000 materialsdiscussed above, the clusters 24 are, respectively, on the order of 1 μmand 2 μm.

In contrast to the PL 4,000 material, which, with respect to its porestructure, is multimodal, a more ideal perfusive particle might comprisea plurality of sets of throughpores and subpores of differing meandiameters. A bimodal pore size distribution can be achieved in suchparticle by mixing equal ratios of particles having two discrete poresizes or by engineering this feature at the polymerization stage.Ideally, mean diameter ratio between throughpore subsets would be lessthan 10, the mean diameter ratio between the smallest throughpore setsand the subpores would be less than 20, and the mean diameter ratiobetween the first pore set, i.e., the intersticies among the particlesmaking up the matrix, and the largest throughpore subset would be lessthan 70, and preferably less than 50. A multimodal material might beproduced by agglomerating 500 Å porons to form approximately 1 μmclusters, which in turn are agglomerated to form 10 μm aggregates, whichin turn may be aggregated to form 100 μm particles. In such a design,the 1 μ m clusters would have intersticies of a mean diameter in thevicinity of a few hundred Å. These would define the subpores and providea very high surface area. Diffusive transport within these pores wouldrarely have to exceed a distance of 0.5 μm or 5,000 Å. Intersticiesamong the 1 μm clusters making up the 10 μm aggregates would permitconvective flow to feed the diffusive pores. These would be on the orderof 0.3 μm in diameter. These throughpores, in turn would be fed bylarger pores defined by intersticies among the 10 μm particles making upthe 100 μm particles. These would have a mean diameter on the order of35 μm.

From the foregoing it should be appreciated that the discussionregarding first and second pore sets and their relative dimensions is anidealization which, although achievable in practice, is not necessarilyoptimal. However, this idealization is useful in understanding thenature and properties of perfusion chromatography systems. In practice,both pore sets can vary widely in average diameter, particularly thesecond pore set.

Referring to FIG. 5C, a different form of perfusive chromatography mediais illustrated as an impervious material 30 comprising a prefabricatedthroughpore 14. As illustrated, the pore comprises a central channel forconvective flow and thin radial fins 32 which extend from the interiorwall 34 of the pore and define a large surface area. At low fluidvelocity, diffusion between the radially directed fins 32 and convectivepore 14 would be required to access the solute interactive surfaces.Higher pressures would effect convective flow within throughpore 14 andaxially within the spaces between radial fins 32, permitting convectivetransport of solutes in close proximity with the solute interactivesurfaces disposed on the walls and fins.

Another form of perfusive matrix (not shown) comprises flow channels,such as relatively uniform pores in a membrane or a hollow fiber, havingadhered to their interior walls fine particles comprising the soluteinteractive surface area. The subpores would be defined by theintersticies among the particles, the second pore set by the flowchannels, and the first pore set by other flow paths disposed, forexample, tangentially to the surface of the membrane, or among hollowfibers in a fiber bundle. Techniques for producing such structures arewell known. The difference between existing structures of this generaltype and one designed for perfusive chromatography lie in the dimensionof the first and second pore sets which are designed as set forth hereinto promote convective flow in both types of channels.

From the foregoing it should be apparent that matrices of the inventionmay be embodies in many specific forms. They may be fabricated frominorganic materials as well as polymers.

OPTIMIZATION OF PERFUSIVE MATRIX MATERIALS

As discussed above, the throughpore Peclet number (P_(e) II) must exceed1 to enter the perfusive domain. However, high PeIIs, at least 5 andmost preferably greater than 10, are preferred. Perfusive behavior alsois dependent on internal surface area. Therefore, it is important thatsubpores or other configurations providing the interactive surface beaccessed readily. As an illustration of the parameters of design of sucha matrix material, it may be instructive to examine the aggregativeformation of particles of the type described above having a given porondiameter.

For a given particle size (D_(p)) the larger the flow channel (d_(p))the fewer flow channels there can be per particle at a constant particlevoid fraction. Furthermore, the larger the flow channel, the larger theclusters have to be to form it, and thus the deeper the diffusivepenetration required to access the surface area. The benefit of usingfewer but larger holes is that perfusion takes effect at relativelylower bed velocities and corresponding pressure drops. Perfusion dependson bed velocity, and the upper limit of velocity is dictated by thepressure tolerance limit of the sorbent particles. At large particlediameters, as illustrated below, this constraint becomes lesssignificant.

FIG. 8 is a graph of bed velocity in cm/hr vs pore diameter in Å for a10 μm nominal diameter particle bed. The graph shows the minimum andmaximum throughpore size to achieve a throughpore Peclet number of 10assuming a 10 μm particle, the diameter of the intraparticle flowchannels is 1/3 of the particle size, and the characteristic porediffusion time is less than convection time. Thus, for example, at 1,000cm/hr, 10 μm particles require a mean pore diameter greater than about5,000 Å in order to achieve a Peclet number of 10 or more. The curvelabeled "maximum pore size" sets forth the maximum mean throughporediameter, for various bed velocities, at which convective flow throughthe pore is so fast that solute diffusion in and out of the subpores istoo slow to permit effective mass transfer to the interactive surfaces.Note that the minimum bed velocity needed to establish perfusion (withP_(e) II>10) diminishes with increasing throughpore mean diameter. Notealso that perfusion will not occur to any significant extent inconventional porous media (<500 Å pore size).

FIG. 9 shows the minimum pore diameter (in thousands of angstroms)needed for various diameter particles (in μm) for various bedvelocities, ranging from 1,000-5,000 cm/hr, required to achieve a Pecletnumber (PeII) greater than 10, making the same assumptions as discussedimmediately above. Clearly, perfusion can be used with larger diameterparticles. For example, a 50 μm particle with 1 μm flow channels,leading to 500 Å diffusive pores, would operate in a perfusive mode atbed velocities exceeding 800 cm/hr.

Analysis of the flow properties of perfusive chromatography matricessuggests that there are very significant advantages to be gained byusing large particles having large throughpores leading to subpores.Where one seeks to maintain resolution, i.e. maintain plate heightconstant, by scaling up a bed having particle size D_(p) 1 andthroughpore size d_(p) 1 then, at constant bed velocity, the size of thelarger particles (D_(p) 2) and their pores (d_(p) 2) is given by theexpression ##EQU5## To scale up at constant plate height and constanttotal pressure drop, the relationship is: ##EQU6## and in general:##EQU7##

As is evident from a study of the foregoing relationships, a linearparticle size/pore size scale-up allows the same separation to beperformed faster and at lower pressure drops. This behavior iscounterintuitive based on current chromatography theory and practice.

To illustrate this scale-up concept, note, from Equation 10, that byincreasing the pore size by a factor 5, the plate height of a 50 μmperfusive particle becomes equal to that of 10 μm perfusive particle at25 times higher velocity and the same pressure drop. In order to operateat a lower pressure drop, the same bed velocity, but with largerparticles, the pore size would have to increase even more to accommodatea constant plate height upon scale-up (see Equation 9). An increase inpore size by about 11 fold is needed to achieve an equivalent resolutionseparation at 25 times lower pressure drop. From Equation 11 it shouldbe apparent that, for example, with 50 μm particles, an increase in bedvelocity of 5 fold, a pressure drop decrease of 5 fold, and a porediameter increase 5 fold will achieve the same resolution faster and atlower pressure drop than for a 10 μm particle.

The table set forth below illustrates these relationships for six casestudies. Column one in each case requires a 5 fold increase in particlesize. In case A, the pore size in the larger particle remain unchangedand the same superficial bed velocity is used (Column 4). In this case,relative to the bed of smaller particles, the larger particle bedoperates at a pressure drop of 1/25th (Column 3) and has a throughporevelocity of 1/25th (Column 5). However, the Peclet number in thethroughpores of the larger particle is only 1/5 that of the smaller, andplate height increases by a factor of 125, greatly decreasingresolution.

In case B, pore size remains constant (Column 2) and superficial bedvelocity is increased by a factor of 5 (Column 4). In this case, thepressure drop is only 1/5, as is the pore velocity. Peclet numberremains constant, but plate height increases by a factor of 25.

In case C, pore size and operating pressure remain constant, resultingin a bed velocity 25 times that of the smaller particle bed. Velocitythrough the pores also remains constant, the Peclet number increases bya factor of 5 and plate height increases by a similar factor.

In case D, the throughpores of the particle are scaled-up by the samefactor as the particle diameter, and pressure drop is maintained,resulting in a 25 fold increase in superficial bed velocity. Thethroughpore fluid velocity therefore increases by a factor of 5, thePeclet number increases by a factor of 25, and plate height remainsconstant.

In case E, the diameter of the throughpores is increased by a factor of125 (5 relative to the particle diameter). Thus, at the same bedvelocities, the pressure drop is 25 times higher than that in thesmaller particle case. Fluid velocity in the throughpores if five timeshigher, the Peclet number increases by a factor of 25, and plate heightstays the same.

Lastly, in case F, where the throughpore is scaled the same way as theparticle size, operating at 5 times the bed velocity one experience only1/5 the operating pressure. Yet the fluid velocity in the throughporesis 5 times that of the base case, the Peclet number is increased by afactor of 25, and plate height, and thus resolution, stay the same.

                  TABLE                                                           ______________________________________                                        1       2       3       4     5     6      7                                  Dp.sub.2                                                                              d.sup.p 2                                                                             ΔP.sub.2                                                                        V.sub.B2                                                                            V.sub.p2                                                                            PeII2  H.sub.2                            Dp.sub.1                                                                              dp.sub.1                                                                              ΔP.sub.1                                                                        V.sub.B1                                                                            V.sub.p1                                                                            PeII1  H.sub. 1                           ______________________________________                                        A   5       1       1/25  1     1/25  1/5    125                              B   5       1       1/5   5     1/5    1     25                               C   5       1       1     25    1      5     5                                D   5       5       1     25    5     25     1                                E   5       125     25    1     5     25     1                                F   5       5       1/5   5     5     25     1                                ______________________________________                                    

In the foregoing analysis, Column 6, showing the ratio of Pecletnumbers, is an indication of the advantage in productivity achieved overdiffusive particles. The plate height ratio is an indication of theadvantage (disadvantage) in resolution achieved over smaller perfusiveparticles. Thus, in cases D, E, and F, very significant increases inthroughput and/or reduced pressure drop are achieved while maintainingthe resolving power of smaller particles.

Accordingly, it is apparent that many trade offs can be made in order tobest utilize the perfusive mode of solute transport in chromatographysystems embodying the invention. It should also be apparent that largeparticles, e.g., greater than about 40 μm in diameter, having largerthroughpores leading to subpores on the order of 300 to 700 angstroms inmean diameter represent a class of matrix materials of great promise.

EXEMPLIFICATION

The advantages of perfusion chromatography have been well demonstratedusing the commercially available particulate media discussed above (PL1,000 and PL 4,000) both untreated and derivatized withpolyethyleneimine, and also with prototype materials manufactured byPolymer Laboratories, Ltd. similar to the PL 4,000 material but having alarger particle diameter. Tests were run using synthetic mixtures ofproteins of the type generally encountered in protein purification andseparation tasks.

Evidence that, unlike conventional chromatography, bandspreading is notexacerbated by high flow rates in the perfusive chromatography realm isset forth in FIG. 10. These chromatograms, prepared by detecting byoptical absorption the protein output of a 50 mm by 4.6 mm column packedwith PL 4,000 material, show quite clearly that the resolution of, forexample, the proteins OVA (ovalbumin) and STI (soybean trypsininhibitor) are similar at 1 ml per minute (350 cm/hr), 2 ml per minute(700 cm/hr), and 4 ml per minute (1400 cm/hr), left to right in FIG.10). These chromatograms achieved, respectively, resolutions of 6.0,6.5, and 6.2.

FIG. 11 shows data illustrative of the ability of perfusivechromatography to produce high resolution very rapid separations ofprotein at high bed velocities and shallow column geometries. FIG. 11was produced with a 5 mm long by 6 mm wide column using PL 4,000material with a flow rate of 3 ml per minute. Note the resolution of thefour test proteins in less than 1 minute.

FIG. 12 compares the performance of nonporous vs perfusive media for theseparation of the test protein mixture. PL 4,000 material (right) isseen to perform in comparable fashion to the nonporous particles (left)in spite of the much larger size of the PL material (10 μm vs. 3 μm).This is in contrast to diffusive media where resolution typically isrelated inversly to the square of the particle diameter.

FIG. 13A through 13D show high resolution separations of 6 proteins inless than 90, 80, 60, and 40 seconds, respectively, at bed velocities of900, 1200, and 1500 cm/hr and 1200 cm/hr respectively, using the PL4,000 underivatized particles (reverse phase). These chromatograms wereproduced on a 6 mm by 5 mm column with a gradient of trifluoroaceticacid and acetonitrile. Chromatogram 13D was achieved by using a steepergradient.

FIG. 14 provides further evidence of the contribution of convectivetransport to perfusive chromatography procedures. It discloses plateheight curves (H vs. flow rate) for lysozyme (A) and acid phosphatase(B) produced using a 250 mm by 4.5 mm column packed with the PL 4,000material eluted with 250 mM NaCl. As illustrated, at low mobile phasevelocity, the plate height curves are indistinguishable fromconventional matrix material. That is, below about 1 ml/min, the plateheight is seen to increase with increase flow rate. However, at highmobile phase velocities, in the range of 1 to 2 ml/min for acidphosphatase, and 2 to 3 ml/min for lysozyme, the plate height curve isactually flat. At very high velocities, i.e., above about 700-800 cm/hrfor acid phosphatase and about 1100 cm/hr for lysozyme, the plate heightrises again, but at a much slower rate than expected for the severediffusion limitations that would prevail under these conditions inconventional media.

FIG. 15A and 15B compare the plate height curves for various linear flowvelocities for, respectively, a diffusively bound polymer bead(Monobeads, Phamarcia) and the PL 4,000 material. Because of pressurelimitations, the Monobeads could not be used at a velocity greater thanabout 1200 cm/hr. At linear velocities as high as 2500 cm/hr,bandspreading with the PL 4,000 particles is less than twice its valueat the minimum. In contrast, by extrapolating this stagnant masstransfer limited regime of FIG. 15A, this value would be nearly fourtimes higher than minimum for the conventional Monobead medium.

One ramification of the enhanced transport kinetics characteristic ofperfusive chromatography is a short cycle time. However, the perfusiveenhancement also can be used to increase resolution with the same cycletime. This is illustrated in FIGS. 16A, 16B, and 16C, chromatogramsshowing the separation of a complex protein mixture using the PL 4,000material. At 350 cm/hr (16A), the procedure is diffusion limited (Pecletnumber in the throughpores less than 1). Separation is fair with a steepgredient of 40 mM CaCl₂ /minute. As shown in FIG. 16B, cycle time can beshortened considerably by increasing the bed flow rate to 4300 cm/hr. Asillustrated in FIG. 16C, by using a bed linear velocity of 4300 cm/hrwith a shallower gradient of 12 mM CaCl₂ /minute, one can obtained amuch higher resolution in a shorter time frame.

FIGS. 17A and 17B show that peak resolution is not affected by anincrease in bed velocity of greater than tenfold for separation of IgGclass 1 and 2. The chromatogram of FIG. 17A was run on a 30 by 2.1 mmcolumn of the PL 4,000 material. The immunoglobulins from mouse asciteswere separated with a 40 mM CaCl₂ /min gradient at a flow rate of,respectively, 0.2 ml/min, and 2.5 ml/min, representing fluid velocitiesof 300 cm/hr and 4300 cm/hr, respectively.

FIGS. 18A through 18F are chromatograms produced by purifyingBeta-Galactosidase from E. coli lysate on the PL 4,000 (A, B, C) and PL1,000 (D, E, F) materials. As illustrated, a full cycle can be performedin less than 15 minutes in the perfusive mode (1200 cm/hr, 18C, 18F)giving essentially the same performances in resolution as obtained in atypical 60 minute cycle 300 cm/hr (18A, 18D). The beta-gal peak isshaded.

FIG. 19A, B, C and D show four chromatograms produced by separating atest protein mixture containing beta lactoglobulin and ovalbumin withthe strong ion exchange versions of the PL 1,000 and PL 4,000 materials.This test mixture was separated with columns packed with the particlenoted and operated at 0.5 and 2.5 ml/min. With 8 micron particles, theseparation is the same at 0.5 ml/min (19A, 19B), and at 2.5 ml/min.(19C, 19D). As shown in FIGS. 19E and 19F, at 5.0 ml/minute, the PL4,000 material performed better than the PL 1,000, since it perfuses toa higher extent. Nevertheless, both separate the mixture adequately.

Conventional liquid chromatography teaching, well corroborated byexperiment, is that "increasing particle size leads to a lowerresolution at a given flow rate, and with increasing flow rates the lossin resolution is increased at a faster rate". However, as noted above,with perfusive matrices, the degree of perfusion is dependent on therelative size of the first and second pore sets, which, for particulatemedia, translate to relative particle diameter and throughpore size.Separation performance in a perfusive regime with large particlestherefore is expected to depend less on flow rate.

The validity of this hypothesis is demonstrated by comparison of FIGS.19 with FIGS. 20. FIGS. 20A and 20B were produced with the proteinsample at 0.5 ml/min using PL 1,000 and PL 4,000 particles,respectively, both having a 20 μm mean particle size. FIGS. 20C and 20Dwere run with PL 1,000 and 4,000 materials, both 20 μm particle size, at2.0 ml/min. FIGS. 20E and 20F were run at 5.0 ml/min using therespective materials. The data show that at 0.5 ml/min, the PL 4,000material performed slightly better than the PL 1,000 material, and, asexpected, when operating at flow rates too low to induce perfusion, bothperform worse than their 8 μm particle size counterparts. At 2.0ml/minute, the gap in performance between these two materials widens,with the PL 1,000 material losing resolving power considerably while thePL 4,000 material can still separate the peaks. At 5.0 ml/minute (1500cm/hr) the 20 μm 4,000 angstrom material can still resolve these peakswhile the PL 1,000 material loses performance almost completely. Thedifference between the performance of the two materials is less at 8 μmthan at 20 μm because, while both perfuse in the smaller particle case(to different extents), with the larger particles the PL 1,000 materialsare expected to perfuse very little in comparison with the 4,000material.

Lastly, FIG. 21 and 22 provide experimental verification of thecalculations discussed above for the difference in breakthroughbehaviors between perfusive particles and conventional porous, diffusivebound particles. FIG. 21, made in a 5 by 50 mm column using Monobeads(Pharmacia) to separate BSA, shows that, at 150 cm/hr, the breakthroughcurve is shaped as expected for a diffusive column operated underequilibrium conditions. As the fluid velocity is increased to 300 cm/hr,deviation from the equilibrium curve begins and at 900 cm/hr prematurebreakthrough is clearly evident. In contrast, as shown in FIG. 22, usingPL 4,000 in the same column to separate the same protein, thebreakthrough curves are essentially equivalent at 300, 1500, and 2700cm/hr.

The invention may be embodied in other specific forms without departingfrom the spirit and essential characteristics thereof. Accordingly,other embodiments are within the following claims.

What is claimed is:
 1. A chromatography method comprising the stepsof:(A) forming a chromatography matrix by packing a multiplicity ofparticles defining throughpores and solute interactive surface regionstherewithin; and (B) passing a fluid mixture of solutes comprisingbiological molecules through said matrix at a velocity sufficient toinduce a convective fluid flow rate through said throughpores greaterthan the rate of solute diffusion through said throughpores and toproduce a Peclet number in a said throughpore greater than 1.0.
 2. Achromatography method comprising the steps of:(A) providing achromatography matrix defining:interconnected first and secondthroughpore sets, the members of said first throughpore set having agreater means diameter than the members of said second throughpore set,and surface regions in fluid communication with the members of saidsecond throughpore set which reversibly interact with a solute, and (B)passing a fluid mixture of solutes comprising biological moleculesthrough said matrix at a rate sufficientto induce convective fluid flowthrough both said throughpore sets and to induce a convective flow ratewithin said second throughpore set greater than the rate of diffusion ofsaid solute within said second throughpore set, thereby to produce aPeclet number in a said second throughpore greater than 1.0.
 3. Themethod of claim 1 or 2 wherein the chromatography matrix defines amultiplicity of subpores comprising said surface regions.
 4. The methodof claim 3 wherein said fluid mixture is passed through said matrix at arate such that the time for said solute to diffuse to and from a saidsurface region from within a member of said second through pore set isno greater than ten times the time for solute to flow convectively pastsaid region.
 5. The method of claim 3 wherein said subpores have a meandiameter less than about 700 Å.
 6. A chromatography method comprisingthe steps ofA. providing a chromatography matrix defining:interconnectedfirst and second throughpore sets, each of which comprises amultiplicity of pores for channeling through said matrix a mixture ofsolutes disposed in a fluid, and surface regions in fluid communicationwith the members of the second throughpore set which sorb a solute insaid mixture B. passing a fluid mixture of solutes comprising biologicalmolecules through said matrix at a fluid flow rate sufficient toproduce:convective fluid flow through both throughpore sets, aconvective fluid flow velocity through said first throughpore setgreater than the fluid flow velocity through the second throughpore set,and a convective fluid flow velocity through said second throughpores etgreater than the diffusion rate of said solute within a member of saidsecond throughpore set whereby the Peclet number therein is greater than1.0, to load solutes from said fluid mixture onto said surface regions,and C. passing an eluant through said matrix to elute a fraction rich ina selected one of said solutes.
 7. The method of claim 6 wherein therelative dimensions of the members of said second throughpore set andsaid surface regions permit flow through the members of said secondthroughpore set at a rate such that the time for a solute to diffuse toand from said surface regions from said second throughpore set iscomparable to or shorter than the time for said solute to flowconvectively past said region.
 8. The method of claim 6 wherein step Bor C is conducted by passing said fluid mixture or eluant through saidmatrix at a bed velocity greater than 1500 cm/hr.
 9. The method of claim6 wherein step B or C is conducted by passing said fluid mixture oreluant through said matrix at a bed velocity greater than 1000 cm/hr.10. The method of claim 6 wherein the step B and C are conducted atfluid flow velocities through the matrix to produce a specificproductivity of at least 1 mg total solute sorbed per ml of matrix perminute.
 11. The method of claim 6 wherein the step B and C are conductedat fluid flow velocities through the matrix to produce a specificproductivity of at least 2 mg total solute sorbed per ml of matrix perminute.
 12. The method of claim 6 wherein the matrix provided in step Acomprises packed particles having a mean diameter greater than 8 μm,said second throughpore set comprises throughpores within the particleshaving a mean diameter greater than 2000 Å, and the ratio of the meandiameter of the particles to the mean diameter of the throughpores isless than
 70. 13. The method of claim 6 wherein the ratio of the meandiameter of the particles to the mean diameter of the throughpores isless than
 50. 14. The method of claim 2 or 6 wherein one of saidthroughpore sets comprise pores having a narrow distribution of porediameters such that 90% of the pores fall within 10% of the mean porediameter.
 15. The method of claim 2 or 6 wherein at least one of saidthroughpore sets comprises a plurality of subsets having differing meansdiameters together producing a wide distribution of pore diameters. 16.The method of claim 1, 2, or 6 comprising the additional step ofcollecting a selected one of said solutes after step B.
 17. The methodof claim 6 wherein said surface regions comprise subpores having a meandiameter less than about 700 Å.
 18. The method of claim 6 wherein thefluid is passed through the matrix in step B or C at a velocity suchthat the Peclet number in the throughpores of the second throughporesset is greater than
 5. 19. The method of claim 6 wherein the Pecletnumber in the throughpores of the second throughpore set is greater than10.
 20. A chromatography method comprising the steps of:(A) providing achromatography matrix defining:interconnected first and secondthroughpore sets, each of which comprise a multiplicity of pores forchanneling through said matrix a mixture of solutes disposed in a fluidand surface regions in fluid communication with the members of saidsecond throughpores set which sorb a solute in said mixture (B) passinga fluid mixture of solutes through said matrix at a fluid flow rategreater than 1000 cm/hr to produce:convective fluid flow through boththroughpore sets a convective flow velocity through said firstthroughpores set greater than the fluid flow velocity through saidsecond throughpore set and a Peclet Number greater than 1.0, and aconvective fluid flow velocity through a member of said secondthroughpore set greater than the diffusion rate of said solute withinsaid member of said second throughpores set, and (C) passing an eluantthrough said matrix to elute a fraction rich in a selected one of saidsolutes.
 21. The method of claim 20 wherein step B or C is conducted bypassing said fluid mixture or eluant through said matrix at a bedvelocity greater than 1500 cm/hr.
 22. The method of claim 20 whereinsufficient solute is used in step B to load solute from said fluidmixture onto said surface regions at a ratio of at least 1 mg totalsolute per ml of matrix per minute.
 23. The method of claim 20 whereinstep B is conducted at fluid flow velocities through the matrixsufficient to produce a specific productivity of at least 2 mg totalprotein sorbed per ml of matrix per minute.
 24. The method of claim 20wherein the matrix provided in step A comprises packed particles havinga mean diameter greater than 8 μm, said second throughpore set comprisesthroughpores within the particles having a mean diameter greater than2000 Å, and the ratio of the mean diameter of the particles to the meandiameters of the pores is less than
 70. 25. The method of claim 20wherein the ratio of the mean diameters of the particles to the meandiameters of the throughpores is less than
 50. 26. The method of claim20 wherein said surface regions comprise subpores having a mean diameterless than about 700 Å.
 27. The method of claim 20 wherein the fluid ispassed through the matrix in step B or C at a velocity such that thePeclet number in the throughpores of the second throughpore set isgreater than
 5. 28. The method of claim 27 wherein the Peclet number inthe throughpores of the second throughpores set is greater than
 10. 29.A chromatography method comprising the steps of:(A) forming achromatography matrix by packing a multiplicity of particles definingthroughpores and solute interactive surface regions therewithin; and (B)passing a fluid mixture including a large biological molecule throughsaid matrix at a velocity sufficient to induce a convective fluid flowrate through said throughpores greater than the rate of moleculediffusion through said throughpores and a Peclet Number greater than1.0.
 30. A chromatography method comprising the steps of:(A) providing achromatography matrix defining:interconnected first and secondthroughpore sets, the members of said first throughpore set having agreater mean diameter then the members of said second throughpore set,and surface regions in fluid communication with the members of saidsecond throughpore set which reversibly interact with a large biologicalmolecule, and (B) passing a fluid mixture of solutes including a saidlarge biological molecule through said matrix at a rate sufficienttoinduce convective fluid flow through both said throughpore sets, toinduce a convective flow rate within said second throughpore set greaterthan the rate of diffusion of said biological molecule within saidsecond throughpore set and to produce in said second throughpore set aPeclet Number greater than 1.0.
 31. A chromatography method comprisingthe steps ofA. providing a chromatography matrix defining:interconnectedfirst and second throughpore sets, each of which comprise a multiplicityof pores for channelling through said matrix a mixture of solutesincluding a large biological molecule disposed in a fluid, and surfaceregions in fluid communication with the members of the secondthroughpore set which sorb a said biological molecule in said mixture B.passing a fluid mixture of solutes including a said biological moleculethrough said matrix at a fluid flow rate sufficient toproduce:convective fluid flow through both throughpore sets, aconvective fluid flow velocity through said first throughpore setgreater than the fluid flow velocity through the second throughpore setand a Peclet Number greater than 1.0, and a convective fluid flowvelocity through a member of said second throughpore set greater thanthe diffusion rate of said biological molecule within said member ofsaid second throughpore set, to load molecules from said fluid mixtureonto said surface regions, and C. passing an eluant through said matrixto elute a fraction rich in a selected one of said biological molecules.32. The method of claim 31 wherein step B or C is conducted by passingsaid fluid mixture or eluant through said matrix at a bed velocitygreater than 1500 cm/hr.
 33. The method of claim 31 wherein steps B or Cis conducted by passing said fluid mixture or eluant through said matrixat a bed velocity greater than 1000 cm/hr.
 34. The method of claim 31wherein steps B and C are conducted at fluid flow velocities through thematrix sufficient to produce a specific productivity of at least 1 mgtotal biological molecule sorbed per ml of matrix per minute.